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Molecular MedicineDOI: 10.1002/anie.200802092

RNA Interference: From Basic Research to TherapeuticApplications

Jens Kurreck*

AngewandteChemie

Keywords:

gene expression molecular biology

RNA interference RNA

Dedicated to Professor Volker A. Erdmann

J. KurreckReviews

1378 www.angewandte.org 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2009, 48, 1378 1398
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1. Introduction

The term RNA interference (RNAi) refers to a cellular

process by which a double-stranded RNA (dsRNA)

sequence-specifically inhibits the expression of a gene. This

very efficient process of posttranscriptional gene silencing

(PTGS), which involves numerous cellular proteins besides

the RNA, is strongly conserved in eukaryotes and presumablyserves as a protection against viruses and genetic instability

arising from mobile genetic elements such as transposons. It

was originally observed in plants,[1] but correctly described for

the first time in the late 1990s for the nematode Caenorhab-

ditis elegans.[2] For this achievement Andrew Fire and Craig

Mello were honored with the 2006 Nobel Prize for Medicine

or Physiology.[3, 4] As measured by the number of publications,

RNAi belongs, along with proteomics, to the most dynamic

fields of biotechnology.[5]

1.1. The Mechanism of RNA Interference

In their key publication, Fire and Mello introduced a long

double-stranded RNA into C. elegans and observed that, as a

result, the expression of the homologous gene was blocked. [2]

Since then, the basic processes involved have been deter-

mined in detail. In a first step, the endonuclease Dicer

processes the long dsRNA into small or short interfering

RNAs (siRNAs) which are around 21 nucleotides long, of

which 19 nucleotides form a helix and 2 nucleotides on each of

the 3 ends are unpaired (Figure 1A). The actual effector ofthe RNAi is the ribonucleoprotein complex RISC (RNA-

induced silencing complex), which is guided by the siRNA to

the complementary target RNA. As a result, the target RNA

is cleaved at a specific site in the center of the duplex,10 nucleotides from the 5 end of the siRNA strand.[6] The

catalytic component that cleaves the target RNA (slicer

activity), has been identified as the protein designated

Argonaut 2 (Ago2).[7] An analysis of its crystal structure

showed that Ago2 contains a domain which resembles

RNase H,[8] a long-known protein that cleaves the RNA

component of a DNA/RNA duplex. After cleavage, the target

RNA lacks those elements which are typically responsible for

stabilizing mRNAs, namely the 5 end cap and the poly-A tail

at the 3 end, so that the cleaved mRNA is rapidly degraded

by RNases and the coded protein can no longer be synthe-

sized.

It is assumed that the loading of the siRNA into the RISC

is accomplished by the RISC-loading complex (RLC), which

consists in Drosophila melanogaster of Dicer-2 and R2D2,

and in mammalian cells of Dicer and the TAR RNA binding

protein (TRBP). Furthermore, it has been shown that during

the activation of the RISC, the strand designated the

passenger (or sense) strand is cleaved, while the other

strand, the guide (or antisense) strand, remains in the

RISC.[10,11] Recent investigations with reconstituted human

RLC demonstrated that Ago2 dissociates from the rest of the

complex after loading with the double-stranded RNA. [12]

1.2. RNA Interference in Mammalian Cells

1.2.1. siRNAs for Targeted Inhibition of Gene Expression

The technique of turning off the expression of specific

genes by dsRNAs could, initially, be applied to a large number

of eukaryotes, such as plants, C. elegans or D. melanogaster,

but could not be applied to mammals since long dsRNAs

trigger an unspecific interferon (INF) response in mammalian

cells. The dsRNA is interpreted by these cells as a pathogen,and protein kinase R is activated, which terminates protein

synthesis in the affected cells.[13] Furthermore, enzymes are

induced which produce 2-5-linked oligoadenylates and

thereby cause an RNase l-dependent unspecific degradationof single-stranded RNA.[14]

Since the INF response is only triggered by dsRNAs which

are longer than 30 nucleotides,[15] the realization that RNAi is

induced by RNAs of approximately 21 nucleotides[6, 16]

[*] Prof. Dr. J. KurreckInstitute of Industrial Genetics, University of StuttgartAllmandring 31, 70569 Stuttgart (Germany)Fax: (+49)711-685 66973E-mail: [email protected]: http://www.uni-stuttgart.de/iig/institut/staff/kurreck/

index.html

Only ten years ago Andrew Fire and Craig Mello were able to showthat double-stranded RNA molecules could inhibit the expression of

homologous genes in eukaryotes. This process, termed RNA interfer-

ence, has developed into a standard method of molecular biology. This

Review provides an overview of the molecular processes involved, with

a particular focus on the posttranscriptional inhibition of gene

expression in mammalian cells, the possible applications in research,and the results of the first clinical studies.

From the Contents

1. Introduction 1379

2. Design and Stabilization of

siRNAs 1382

3. Vector Expression of shRNAs 1384

4. Unspecific Side Effects 1386

5. Delivery 1387

6. Applications of RNA

Interference 1391

7. Summary and Outlook 1395

RNA InterferenceAngewandte

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provided a solution to the problem: With their ground-

breaking work that chemically synthesized 21-mer siRNAs

trigger RNAi in mammalian cells, Tuschl and co-workers

opened the way to use RNAi for experiments in mammalian

cells.[17] This created new opportunities, not only for research,

but also for therapeutic treatments. The presynthesized

siRNA is phosphorylated on its 5 end by the kinase Clp1

after entering the cells[18] which is then followed by the RNAipathway described above (Figure 1 B).

RNAi expanded the repertoire of the already well known

oligonucleotide-based strategies of PTGS. Antisense oligo-

nucleotides have been employed for the last 30 years to

inhibit the expression of genes at the mRNA level. Antisense

and RNAi strategies have many things in common, such as the

necessity to identify suitable binding sequences on the targetRNA, the stabilization of the oligonucleotide by chemical

modification, or the transport of the negatively charged

polymer across the cell membrane. Experience in the

antisense field allowed for very rapid progress to be made

with the new RNAi strategy.[19] There are, however, important

differences between the two technologies: Antisense oligo-

nucleotides are single-stranded (modified) DNA molecules,which primarily induce the cleavage of the target RNA in the

cell nucleus by activation of RNase H. In contrast, RNAi is

triggered by double-stranded RNA, which functions primarily

in the cytoplasm. Ago2, the most important component of the

RISC, is localized in the p bodies.[20] As a result, the central

steps of RNAi appear to take place in these discrete

structures of the cytoplasm. In the case of RNAi, an

endogenous cellular pathway is followed, which could explain

the high efficiency with which siRNAs are able to inhibit the

expression of their target genes. They can be up to 1000 timesas efficient as traditional antisense oligonucleotides against

the same target molecule.[21,22] While no particularly impor-

tant region could be determined for the normally 1520

nucleotide long antisense oligonucleotides, the seed region

(positions 28 of the antisense strand, Figure 1A) is of great

importance for siRNAs, since it is presumably here that the

interaction with the target RNA begins.

The effects of siRNAs are transient. The degradation of

the target RNA usually begins immediately after the siRNA

enters the cell; however, the decrease in the amount of

protein depends on the half-life of the target protein.

Normally a pronounced inhibitory effect can be observed in

cell culture within 48 h of transfection of an siRNA; however,there are proteins with a very slow rate of turnover, which can

be stable for much longer. Also one must keep in mind that inmost cases the target gene is not completely shut off, which is

why RNAi is referred to as a knockdown technology as

opposed to knockout in the case of transgene animals created

by homologous recombination.

The inhibition of the expression of the target gene usually

lasts for five to seven days both in vitro[23] and in vivo.[24]

Interestingly an siRNA can work for different lengths of timein different species: An siRNA against apolipoprotein B was

active in mice for only a few days and after nine days was back

to 70 % of its initial starting level, whereas the knockdown in

nonhuman primates was still effective after 11 days.[25]

Theduration of action of an siRNA presumably depends on

numerous factors, such as the target organ, the target gene,

and the species. Intracellularly expressed short hairpin RNAs

(shRNAs) can be used instead of chemically synthesized

siRNA to extend gene silencing (see Figure 1 B and Sec-

tion 3).

RNAi is primarily a process of PTGS, that is, gene

expression is inhibited by a selective blockade of an mRNA. It

has also been reported that RNAi can alter the chromatin

structure in the nucleus and thereby influence transcrip-

tion.[26] This has been observed in particular for yeast, plants,

and fruit flies. The importance of RNAi for transcriptional

Jens Kurreck studied biochemistry and phi-losophy at the Free University (FU) of Berlinand received his doctorate in 1998 at theTechnical University of Berlin. After a stay atArizona State University he went to the FUBerlin, where he completed his habilitationin 2006. Since 2007 he has been Professorfor Nucleic Acid Technology at the Universityof Stuttgart. His work involves the applica-tion of RNA interference for medically rele-vant topics, in particular virology and painresearch.

Figure 1. A) Structure of an siRNA. The two strands of the siRNA forman approximately 19 nucleotide duplex. Two nucleotides hang overfrom each 3 end. Deoxythymidine is often used as the overhangs inchemically synthesized siRNAs. The position at which the complemen-tary target RNA is cleaved is indicated with an arrow, and the seedregion, through which the interaction with the target RNA begins, isindicated. B) Simplified model of the RNAi mechanism in mammaliancells. After uptake of the chemically synthesized siRNAs into the cells,they are loaded onto the RISC by the RLC, in the course of which thesense strand is removed. The antisense strand guides the RISC to thecomplementary target RNA, which is cleaved by the Ago2 protein. Alonger term inhibition of gene expression can be accomplished whenan shRNA is expressed intracellularly instead of by the exogenousapplication of an siRNA. (Figure adapted from Ref. [9].)

J. KurreckReviews

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gene silencing in mammals has, in contrast, not yet been

clearly demonstrated.

1.2.2. Endogenous Short RNAs: miRNAs, piRNAs, and esiRNAs

Besides the previously described siRNAs, which can be

employed as research tools and potential therapeutics for the

artificial regulation of gene expression, the importance of

endogenous short RNAs which do not code for proteins is

becoming increasing clear. The role of the approximately 21

nucleotide long micro-RNAs (miRNAs) in the posttranscrip-tional regulation of genes has been investigated very inten-

sively in the last few years.[27] In Version 11.0 of the miRBase

data base (http://microrna.sanger.ac.uk) from April 2008

there are over 6000 miRNAs listed from animals, plants,

and viruses; for humans alone over 1000 miRNAs are

predicted.

In the nucleus, miRNA precursors (pri-miRNAs) are

formed by special miRNA genes or as introns from protein-

coding polymerase II transcripts. They are processed by the

RNase III Drosha to approximately 70 nucleotide long pre-miRNAs, which are transported out of the nucleus by

Exportin-5 and are cleaved there by Dicer to become the

functional miRNAs (Figure 2). Similar to siRNAs, they also

form a ribonucleoprotein complex with Argonaut proteins

and bind to their target RNAs. However miRNAs preferen-

tially recognize target sequences in the 3-untranslated region

(3-UTR) of mRNAs, and binding often takes place with an

incomplete homology, although a perfect base pairing in the

previously mentioned seed region (positions 28 of the

miRNA) forms the core of the interaction. Depending on

the degree of homology between the miRNA and mRNA, the

result can be an irreversible cleavage of the target molecule or

merely repression of translation.The precise mechanism of the miRNA-dependent post-

translational repression of gene expression is currently thesubject of intense research.[27] According to the two most

important models, either translation is blocked or the mRNA

is destabilized. The inhibition of translation could take place

at the level of initiation. In this process it is assumed that the

Ago2/miRNA complex interacts with the cap structure at the

5 end after binding to the 3-UTR of an mRNA and thereby

prevents the binding of the initiation factor eIF4E. As aresult, the initiation complex cannot be formed. Alternatively,

translation could be slowed after initiation or the ribosomes

could dissociate prematurely. According to the alternative

model, the mRNA is de-adenylated by the miRNA, whichmakes 3!5 degradation possible or the cap is removed,

which would enable degradation in the 5!3 direction by

exonucleases. Possibly there are other mechanisms through

which miRNAs could work.

It is assumed that miRNAs control the activity of about

30% of all protein-coding genes in mammals. Since every

miRNA regulates numerous mRNAs, and in turn mRNAs can

be influenced by numerous miRNAs, this results in an

extremely complex regulatory network. So it is hardly

surprising that miRNAs are involved in all cellular processes

that have been investigated and play an important role in

numerous diseases, such as cancer,[28] viral infections,[28, 29] and

genetic diseases.[30] For further Information concerning the

activity and function of miRNAs the reader is referred to

recent review articles.[27, 31]

A further class of short regulatory RNAs are associated

with Piwi proteins and are thus referred to as piRNAs.[32]

These RNAs, at around 2430 nucleotides in length, are

slightly longer than typical siRNAs or miRNAs. They are

presumably processed from single-stranded precursors and

are found principally in germ cells. Besides their importancein the control of mobile genetic elements, a function in

spermatogenesis is also suspected.

Recently, endogenous siRNAs (esiRNAs) were found by

comprehensive sequencing of short RNAs in mammalian cells

(mouse oocytes).[33, 34] It was previously assumed that an

RNA-dependent RNA polymerase activity was required for

the production of esiRNAs, but this is not found in mammals.

It has now been shown that other double-stranded RNAs,

such as long hairpin structures or complementary sequences,

can serve as the starting point for the production of esiRNAs.

The esiRNAs derive from retro-transposons and apparently

function as their inhibitors. In addition, esiRNAs from

Figure 2. miRNA pathways in mammalian cells. RNAs are transcribedin the nucleus in the form of a precursor (pri-miRNA), which isprocessed by the RNase III Drosha to pre-miRNA. In this process, theDrosha complexes with the DGCR8 protein. The pre-miRNA isexported out of the nucleus and into the cytoplasm by Exportin-5 andcleaved there by Dicer (complexed with TRBP) to form the functionalmiRNA, which in turn combines with an Argonaut protein (Ago) toform an miRNAribonucleoprotein (miRNP) complex. The miRNA can

either cause endonucleolytic cleavage of the target mRNA throughAgo2 or block translation in the case of partial complementarity.(Figure adapted from Ref. [27].)


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pseudogenes have been found, which could be of significance

for the regulation of protein-coding transcripts.

2. Design and Stabilization of siRNAs

2.1. Design of siRNAs

The first important step for the successful application of

RNAi is the design of efficient siRNAs. The original

assumption, that it is not necessary to search for suitabletarget sequences in the target RNA,[35] proved to be too

optimistic. In practice, the efficiency of different siRNAs

against the same target RNA varies drastically.[36] Apparently

factors intrinsic to the siRNA itself and the characteristics of

the target RNA both play a role in silencing.[37]

The probability of identifying a very efficient siRNA was

significantly increased after it was discovered that both

strands of an siRNA or miRNA are not equally likely to be

incorporated into a RISC. Instead, the strand with a lower

thermodynamic stability (namely, a higher A/T content) at its5 end is preferred.[38, 39] Thereafter, the molecular basis for

this strand asymmetry could be determined.[40] In D. mela-

nogaster, RISC is loaded by a heterodimer of Dicer-2 and the

dsRNA-binding protein R2D2. Here, R2D2 binds to the more

thermodynamically stable end of the double-stranded RNA

and thus determines which strand associates with the RISC as

the guide strand. In a detailed study with 180 siRNAs against

two different RNA targets, besides the relative stability of the

two ends, additional criteria (preference for special bases in

certain positions) were identified which are common among

the functional siRNAs.[41]

In these experiments, however, the significance of each

parameter was determined independently of one another. Toalso take into account synergistic influences of multiple linked

parameters, an artificial neuronal network was trained with a

dataset of over 2000 siRNAs against 34 different mRNAs

(BIOPREDsi-algorithm).[42]

An extensive survey of the activity of published siRNAs

has shown, however, that there are a number of very active

siRNAs which do not correspond to the proposed criteria,

while numerous other carefully designed siRNAs are inactive.

Recently, even the hypothesis that the relative stability of thetwo ends has an influence on their efficiency has been called

into question.[43] Neither in an experimentally investigated set

of different siRNAs nor in a comprehensive analysis of

published siRNAs or siRNAs posted to databanks could acorrelation between the terminal stability of the siRNA and

its silencing activity be found. Other characteristics of the

siRNA also possibly play a role. For example, it has been

shown that siRNAs whose antisense strands form stable

helices at their ends only show a low level of activity.[44] The

authors advise, therefore, designing siRNAs such that the

antisense strand is as unstructured as possible.

Besides the siRNA itself, the target RNA could also play

an important role in silencing. This could help to explain why

the expression of some targets is easily inhibited, while the

knockdown of others is more difficult. In a study with several

thousand siRNAs, which were conceived for different genes

according to the BIOPREDsi algorithm, 70% of the inves-

tigated kinase genes were easy silenced (defined as two of two

tested siRNAs working), while 6 % of the genes could not be

down-regulated by up to 10 different siRNAs.[45]

Studies with antisense oligonucleotides have already

shown that the accessibility of the binding region on the

target RNA of oligonucleotides is of great importance for the

efficiency of silencing. A correspondence between the

accessibility for antisense oligonucleotides and siRNAs has

been demonstrated.[46] In a more comprehensive analysis, the

accessibility of target RNAs was predicted by an iterativebioinformatic approach and by experimental RNase H map-

ping.[47] The results showed that siRNAs against predicted

highly accessible areas were more efficient than those whose

target sequence was inaccessible. The relative thermodynamic

stability of the two ends of the siRNA proved, in contrast, not

to be a suitable criterion for the prediction of the efficiency of

an siRNA.

We analyzed the influence on silencing of the thermody-

namic design of the siRNA and the accessibility of the target

RNA more closely with the help of artificial target struc-tures.[48] We were able to confirm in reporter assays the strand

asymmetry, namely, that the target sequences in the natural

orientation led to a stronger silencing than the other way

around. On the other hand, there was a clear correlation

between the local free energy of the siRNA binding region

and silencing. We therefore proposed a two-step model to

describe the inhibitory efficiency of siRNAs (Figure 3):

Initially the thermodynamic characteristics of the siRNA,

that is, the relative stability of the two ends, determine the

asymmetric incorporation of the two strands into the RISC. In

a second step the accessibility of the binding region of the

Figure 3. Two-step model to explain the efficiency of siRNA (s: sensestrand, as: antisense strand): 1) Depending on the relative stability ofthe two ends of an siRNA, one of the two strands is preferentiallyassembled into the RISC. The retention of the strand complementaryto the target RNA can be achieved through the selection of a suitablesequence. 2) An antisense strand assembled into the RISC can,however, be unsuitable for silencing when the complementarysequence of the target RNA is inaccessible. The local structure of thetarget region thus also influences silencing significantly. (Figureadapted from Ref. [48] with permission from Elsevier.)

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siRNA on the target RNA affects the strength of the

silencing. This model was confirmed in an analysis of

around 200 siRNAs and shRNAs against over 100 different

human genes.[49] According to this study, the accessibility ofthe target RNA for the siRNA is of greater importance than

the duplex asymmetry for efficient knockdown. In a further

report it was shown that the accessibility of the 3 end of the

target RNA is particularly important.[50] As already men-

tioned in Section 1, the interaction between the siRNA or

miRNA and the target RNA begins in the seed region.

Some algorithms for the design of siRNAs, such as theSfold web server,[51] take into account not only the thermody-

namic characteristics of the duplex but also the predicted

secondary structure of the target RNA. It must be emphasized

that none of the models proposed so far can guarantee a

successful prediction of the activity of an siRNA. There must,

therefore, be other factors which still need to be identified, in

particular synergistic effects, which influence the efficiency of

RNAi experiments.

Conventional siRNAs consist of a 19-mer duplex and two

nucleotide long overhangs on each of the 3

ends. It has,however, been reported that longer siRNAs can be more

efficient. In an experiment with siRNAs of various lengths,

27 mers had an efficiency up to 100 times higher than the

conventional 21 mers.[52] In a further study, 29-mer shRNAs

were proven to be especially potent.[53] The long duplexes

were initially processed to 21 mers by Dicer and were thus

presumably more efficiently assembled into the RISC by the

RLC.

The problem of the design of individual siRNAs can be

bypassed by the use of enzymatically synthesized siRNA

pools.[54] First, long dsRNAs are generated which can be

processed with bacterially synthesized RNase III or recombi-

nant Dicer to endoribonuclease-prepared siRNAs. This mixof siRNAs can harbor the risk of increased off-target effects

(see Section 4); on the other hand, each individual siRNA is

present at a very low concentration so that the undesirable

side effects are apparently diluted out. With this method,

inexpensive comprehensive libraries against the complete

human and mouse genome have been manufactured.

2.2. Chemical Modification of siRNAs

Although unmodified siRNAs can be used in cell cultures,

it can be advantageous to build modified nucleotides into the

siRNA so as to specifically inhibit the expression of a gene.The primary reason for the chemical modification of siRNAs

is the increase in resistance to nucleolytic degradation. In fact,

although siRNAs have an unexpectedly long life, it is usually

necessary to stabilize them further by the use of modified

nucleotides for in vivo applications. Modifications can often

lengthen the half-life of the siRNA in plasma and improve its

pharmacokinetic characteristics. Furthermore, new function-

alities can be introduced, fluorescent markers or lipophilic

groups, for example, which improve cellular uptake. The rapid

successful incorporation of chemically modified components

can be attributed to the experience gained in the field of

antisense technologies. A multitude of modified nucleotides

have been employed in the past years for siRNAs, of which

several selected examples will be explained here. Further

details have been explained extensively in comprehensive

review articles.[5557]

The incorporation of unnatural nucleotides into siRNAs

presents a particular challenge, since the modifications must

not affect the silencing activity of the siRNA. In this context it

is important to remember that the two strands of the siRNA

have different functions: while the guide strand is assembled

into the RISC and leads the complex to the target RNA, the

passenger strand is discarded in loading the RISC. Thepassenger strand is, therefore, more likely to tolerate modi-

fications, but the guide strand can also have modified

nucleotides built into suitable positions.

Of particular importance is the hydroxy group at the

5 end of the guide strand, which must be phosphorylated for

entry of the siRNA into the RNAi pathway. Correspondingly,

an siRNA whose 5 end is blockedfor example, by an amino

linkerloses its inhibitory activity.[58] Comparatively simple,

in contrast, is the incorporation of functional groups on the

ends of the passenger strand. In this way it is possible to followthe localization of an siRNAwith a fluorophore on the 5 end

of the passenger strand without having a grave influence on its

silencing activity.[59] Furthermore, the cellular penetration of

the siRNA can be improved with a lipophilic component such

as 12-hydroxylauryl acid or cholesterol (see also Sec-

tion 5.1.1).[60]

The most common modification for the stabilization of

antisense oligonucleotides is phosphorothioate DNA, in

which an unlinked oxygen atom is substituted by a sulfur

atom. Phosphorothioates are very stable with respect to

nucleases and are comparatively simple to manufacture. RNA

variants of the phosphorothioates (Figure 4) have therefore

also been built into siRNAs. These modifications are funda-mentally tolerated by the RNAi machinery; however, toxic

side effects have been observed when the phosphorothioate

content is high.[61]

Nucleotides have also been used whose ribose was

modified at the 2-position, for example, 2-O-methyl RNA

Figure 4. Selected modified nucleotides which can be employed tostabilize siRNAs.


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and 2-fluoro-modified nucleotides (Figure 4). The fluoro

substituent is very small, and does not seriously influence the

functionality of the siRNA.[6163] The significantly larger

methyl group, in contrast, inhibits the RNAi function whenthe entire siRNA consists of 2-O-methyl-substituted nucleo-

tides.[63] Therefore, modification types are sought which

increase the stability of siRNA without reducing their

silencing activity. Blunt-ended siRNAs proved to be suitable

when the RNA units and 2-O-methyl nucleotides alternate in

both strands, so that a modified nucleotide is opposite an

unmodified one.[64] Such modified siRNAs were injected intomice as components of lipoplexes (see Section 5.1.1).[65] The

siRNAs were taken up by vascular endothelial cells and

reduced the level of the target mRNA and of the target

protein.

A further modification commonly used in past years is the

locked nucleic acid (LNA, Figure 4).[6668] LNAs have numer-

ous desirable characteristics such as high nuclease stability

and high affinity for the target structure; their incorporation

into an RNA duplex, however, causes serious structural

changes. A complete modification of an siRNA with LNAs istherefore impossible, but a few LNA monomers can be built

into the siRNA without loss of its silencing ability. [62] In a

systematic study, the positions of the antisense strand were

identified which tolerate the substitution of the RNA

nucleotides by an LNA component without loss of activity.[69]

The incorporation of LNAs into siRNAs not only increases

the nuclease stability, it can also reduce the off-target effects

of an siRNA (see Section 4) by inactivating the sense strand

and increase the efficiency of siRNAs by improved loading of

the RISC. Corresponding LNA-modified siRNAs showed

favorable characteristics in systemic use in vivo compared to

unmodified siRNAs.[70]

We used the method of inactivating a strand of an siRNAby the incorporation of LNAs to analyze in detail the

mechanism of RNAi-induced inhibition of the coxsackie

virus.[71] These cardiotropic viruses, which belong to the family

of the Picornaviridae, possess a single positive-stranded

genome, from which during replication a negative strand is

copied as an intermediate. The selective inactivation of one of

the two strands by LNAs showed that only siRNAs against

the genomic positive strand possess an antiviral activity.

In a further study, a triple-stranded siRNA construct wasemployed, in which the antisense strand was hybridized with

two shorter 1012 nucleotide long complementary strands.[72]

These so-called small internally segmented interfering RNAs

(sisiRNA) were modified at various positions with LNAs andhad a very high serum stability and silencing activity.

The fact that the various modifications in different

positions of the siRNA could be built into the siRNAwithout

a drastic loss of activity suggested the possibility of combining

various types of RNA analogues. In this way all of the OH

groups of an siRNA could be substituted successfully: all the

pyrimidines were replaced by 2-fluoro-modified nucleotides,

the purines of the sense strand by deoxyribonucleotides, while

2-O-methyl-RNAs were used for the purines of the antisense

strand.[73] Furthermore, the ends were protected by inverted

abasic sugars and a phosphorothioate bond. These completely

modified siRNAs had a half-life in human serum of several

days, as opposed to several minutes for their unmodified

forms, and were significantly more efficient than the starting

siRNA in a vector-based in vivo model of hepatitis B virus

(HBV) infection.

3. Vector Expression of shRNAs

A great disadvantage of chemically synthesized siRNAs is

that their activity is transient and only lasts several days,

because the siRNAs degrade over time and are diluted out bycell division. It was, therefore, a major advance when in 2002

several research groups simultaneously developed expression

systems in which the siRNA is continuously generated in

cells.[74]

3.1. Expression Plasmids for shRNAs

In the most common system the siRNA is converted into a

DNA sequence which codes for the sense strand, a loop, andthe antisense strand. This DNA template is transcribed from a

vector under the control of polymerase III promoters. These

promoters are optimized for the generation of large amounts

of precisely defined RNAs. The most commonly used are the

promoter of the U6 component of the spliceosome as well as

the H1 promoter of the RNA component of RNase P. During

transcription, a self-complementary RNA is created, which is

referred to as an shRNA. The shRNA is processed intra-

cellularly by Dicer into siRNA, which mediates silencing.

The shRNA expression systems led to the creation of new

applications for RNAi. Usually a vector-expressed shRNA

works significantly longer than chemically synthesized

siRNA. Plasmids equipped with a resistance gene can beused to select transfected cell lines in which the target gene

can be down-regulated for several months.[75]

In addition, transgenic animals can be generated in which

the gene of interest is permanently inhibited by using shRNA

expression vectors. For example, the shRNA expression

cassette can be incorporated into embryonic mouse stem

cells by electroporation[76] or lentiviral transfer[77] (see Sec-

tion 5.2.1). A problem of this method is that integration of the

transgene is random, so the silencing efficiency can varyconsiderably depending on the integration site. Furthermore,

important cellular genes can be destroyed. For this reason a

locus was sought that guaranteed a strong and predictable

shRNA expression. The Rosa 26 locus fulfils these require-ments and is used to integrate the transgene homologously by

recombinase-mediate cassette exchange (RMCE). The

knockdown was 8095% when there was a single copy of

the shRNA-expression cassette in most analyzed organs.[78]

An advantage of this procedure relative to conventional

knockout techniques is the immense saving in time: The

shRNA-expressing animals are available for investigation in

around three to four months, while with knockout animals

back-crosses that can take up to several years are often

necessary before the gene can be deleted from both chromo-

somes in a genetically defined background.

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Surprisingly, we have recently observed phenotypic differ-

ences between knockout and shRNA-expressing animals.[79]

In this study, the function of the vanilloid receptor TRPV1,

which plays an important role in pain perception, wasinvestigated in detail. While the reaction of the shRNA-

expressing animals was in accordance with published data

from TRPV1-knockout animals in most tests, such as

capsaicin-induced hypothermia and colitis, and their reaction

to a heat stimulus, they showed pronounced differences in the

perception of neuropathic pain. While the knockout of

TRPV1 had no impact on the perception of neuropathicpain, the mechanical hypersensitivity and allodynia in the

shRNA-expressing animals was significantly reduced in

comparison to wild-type animals. This finding agrees with

results from small molecule receptor antagonists.[80] The cause

for the differences in the behavior of knockout and shRNA-

expressing animals is not yet fully understood; however, a

complete knockout and a partial knockdown appear to lead to

differences in compensation mechanisms. One should keep in

mind that small molecule pharmacological substances also

only partially inhibit their targets, so that the partial knock-down in an RNAi experiment may better reflect the outcome

of a medicinal therapy with substances directed against that

target.

A further advantage of the RNAi technology is its broad

applicability. While classical knockouts by homologous

recombination are only routinely done with mice, shRNA

vector technology allows genes to be turned off in other

species, such as rats.[81] A further development of this idea is

the creation of disease-resistant domestic animals with the

help of RNAi. In goat foetuses and bovine blastocysts, RNAi

shut off the prion protein (PrP), which aggregates in trans-

missible spongiform encephalopathy (TSE).[82] In this way it

was possible to generate domestic animals which are resistantto BSE and related diseases. Cattle could be made resistant to

foot-and-mouth disease by using a similar method. The

creation of transgenic domestic animals, however, not only

results in technological challenges, but also has ethical and

social implications which must not be neglected.

3.2. MicroRNA-Type shRNAs

While the shRNAs in the systems described so far are

expressed under the control of polymerase III promoters,

modern systems can also employ polymerase II promoters.

This results in transcripts with a cap at the 5 end and a poly-Atail at the 3 end, which are not compatible with the RNAi

machinery. Nevertheless, to use polymerase II promoters, the

expression of miRNAs is simulated. These alternatives are

usually components of longer pre-mRNAs which are tran-

scribed under the control of polymerase II promoters. A

naturally occurring miRNA can be replaced with an artificial

shRNA in the sequence context of the miRNA.[83] The RNA

polymerase II first generates a long primary transcript, from

which Drosha cuts out the pre-miRNAs. These are exported

into the cytoplasm where Dicer processes them into siRNAs,

which are assembled into RISC.

Comparative studies with conventional and miRNA-type

shRNAs against HIV have shown that the latter are up to

80% more efficient.[84] Besides their high efficiency, the

miRNA-type shRNAs have other advantages relative toclassical shRNAs. For one thing, they allow the simultaneous

expression of a protein-coding sequence upstream of the

miRNA segment. In this way, a reporter such as GFP or a

relevant functional protein can be expressed with the shRNA

at the same time. Secondly, polycistronic expression becomes

possible, that is, more than one microRNA-type shRNA can

be expressed at the same time from a single transcript.[85] Inthis manner, either several genes can be silenced at once or a

target gene can be very efficiently inhibited by several

shRNAs. A third advantage is the option of using cell-type-

specific promoters. While polymerase III promoters mediate

a strong and ubiquitous expression, there are a large number

of different polymerase II promoters which are only active

under certain conditions or in specific cell types. For example,

an miRNA-type shRNA against the transcription factor

Wilms Tumor 1 was expressed under the control of the

proximal promoter of the murine gene Rhox5. This specifi-cally inhibited the expression of the target gene in nurse cells

of the testis.[86]

3.3. Inducible Systems

The vector systems also provided the opportunity to

regulate RNAi by pharmacological substances. These systems

may be differentiated into reversible and irreversible types. In

reversible systems, expression of the shRNA is turned on by

the addition of an inducer. When the inducer is taken away,

transcription of the shRNA ceases and the target gene of the

siRNA is once again expressed. In irreversible systems,shRNA expression can be induced, but cannot be turned off

again. This form of regulation is widely employed when genes

which are essential for embryonic development are to be

investigated in adult organisms.

The most common reversible shRNA expression system is

based on tetracycline (tet) controlled transcription.[87] For tet

control, the promoter is usually modified by the addition of a

tet operon, to which a repressor protein binds. The addition of

an inducersuch as tetracycline or its more commonly usedstructural analogue doxycycline (dox)results in a structural

change in the tet repressor being induced such that it is

released from the tet operon, which opens the way for

transcription of the shRNA.The tet system functions in vitro as well as in vivo. For

example, an shRNA against the polo-like kinase 1 (Plk1) was

dox-dependently expressed to study the importance of the

target gene for the proliferation of cancer cells.[88] It was

shown by inoculating these cells into immunodeficient nude

mice that the RNAi-mediated silencing could be modulated

in a dox-dependent manner in vivo. In a further study,

transgenic animals were generated according to the previ-

ously described RMCE procedure, in which the shRNA

expression could be reversibly induced by dox. [89] In this way,

the target gene, which codes for the insulin receptor, could be

down-regulated for a chosen period of time.


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The tet system combines numerous advantages: It has a

low background activity in the absence of an inducer, is

strongly inducible, and quickly reversible after removal of the

inducer, and the inducers tetracycline and doxycycline arenontoxic, well-characterized pharmacological substances.

Besides the described system, there are numerous other

variants of tet control and other reversible regulation systems,

which are explained in a recent review article.[90]

The cre-lox system has been widely used for many years

for conventional knockouts and has also been employed as an

irreversible method for conditional RNAi. In this system,transcription of the functional shRNA is destroyed by the

insertion of an additional DNA segment into the expression

cassette. For example, a neomycin (neo) resistance gene

flanked by two loxPsites can be integrated into the shRNA-

coding region.[91] CRE recombinase removes the interrupting

sequence when expressed in the same cell and induces the

synthesis of the shRNA. Alternatively, the stuffer sequence

can also be inserted in the promoter region. Cre-lox systems

allow temporal control of RNAi suppression, for example,

induction after embryonic development as well as tissue-specific silencing when CRE recombinase is expressed in

certain cell types.

4. Unspecific Side Effects

4.1. Off-Target Effects

Small molecular pharmacological substances which typi-

cally bind to proteins and inhibit their catalytic cores or block

membrane-bound receptors usually bind to their target

molecules through spatial interactions. This often results in

undesirable side effects when the substance also binds toother structurally similar proteins. Since RNAi applications

are based on WatsonCrick base pairing between an oligo-

nucleotide and an RNA, there was hope that undesired side

effects played no role when a target sequence that only

appears once in the genome was used. In practice, a single

mismatch can lead to a complete loss of silencing.[75, 92]

More extensive microarray analyses, with which global

profiles of gene expression can be created, showed, however,

that siRNAs are not completely specific. While initial studiessuggested that the so-called off-target effects of siRNAs are

dose-dependent and can be avoided by the use of lower

concentrations of siRNA,[93] other studies showed that the

unspecific effects have a similar dose response to the intendedknockdown of the target gene.[94] The identity of as few as

eleven nucleotides between the antisense strand of the siRNA

and an mRNA can result in the down-regulation of an mRNA

which is not the intended target. These off-target effects can

have effects on the phenotype, for example, the viability of

cells.[95]

More recent investigations have shown that it is not the

overall identity of an mRNA with the siRNA, but rather the

perfect correspondence between parts of the 3-UTR and the

seed region (positions 27 or 2-8) of the antisense strand of

the siRNA which determines whether gene expression is

influenced (Figure 5).[96] In a systematic study the frequency

of all 4096 possible hexamers in the 3-UTR of the tran-

scriptome was investigated.[97] It was shown that some

hexamers are rare while others are considerably more

common. It became clear in a microarray analysis that

siRNAs with common seed regions trigger stronger off-

target effects than those for which there are only a few

complementary sequences. This means that off-target effects

can be reduced by clever design of the siRNA. Furthermore,

the specificity of siRNAs can be reduced through the

incorporation of modified nucleotides. It is comparativelyeasy to completely inactivate the sense strand by modifica-

tions so that the danger of off-target regulation can be

reduced to a minimum. Changes to the antisense strand are,

on the other hand, more challenging since the inhibitory

effects on the expression of the target gene must not be

influenced. A single 2-O-methyl substitution on the ribose of

the second nucleotide was shown to be enough to significantly

reduce off-target effects while maintaining silencing activity

(Figure 5).[98]

4.2. Interferon (INF) Response

Besides the regulation of partially homologous mRNAs,

siRNAs can surprisingly also trigger an interferon (INF)

response, although it was originally assumed that these

responses are only induced by double-stranded RNA mole-

cules greater than 30 nucleotides in length. A complete

analysis of the INF-stimulated genes revealed, however, that

siRNAs can also activate the interferon system, presumably

mediated by protein kinase R.[99] This effect is not specific for

siRNAs, but has also been observed for vector-expressedshRNAs.[100]

Presumably the Toll-like receptors (TLR) and the heli-

cases RIG-1 and Mda5, in addition to protein kinase R, also

play an important role in the recognition of siRNAs by theimmune system. Three members of the TLR family recognize

RNA and can trigger an immune response through a complex

signaling pathway (Figure 6). It could be shown for plasma-

cytoid dendritic cells that siRNAs induce INF-a via TLR7.[101]

The activating effects of the siRNAs on endosomal TLRs is

dependent on the sequence of the siRNA.[102] As a result,

motifs could be identified which led to a strong induction of

the immune response. This means that immunostimulation

can be circumvented by avoiding the use of these motives in

an siRNA. For special applications, such as the treatment of

viral infections or cancer, strongly immunomodulatory

siRNAs which have two functions, knockdown of the target

Figure 5. Base pairing between nucleotides 28 of the siRNA (seedregion) and mRNAs can lead to off-target effects in RNAi applications.These undesired side effects can be significantly reduced by a 2 -O-

methyl substitution of the second nucleotide (circled).

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gene and induction of interferons, could be used deliber-

ately.[103]

In a recent publication it was reported that unspecific

effects of siRNAs can also be mediated by TLR3. The

investigation of the anti-angiogenetic effects of siRNAs,

which are used, for example, in the treatment of age-related

macular degeneration (see Section 6.3.1), showed in an

animal model that unspecific siRNAs without homologous

sequences in the mammalian genome were just as efficient assiRNA against the vascular endothelial growth factor

(VEGF) or its receptor.[105] These effects were not dependent

on a sequence-specific silencing of the target, nor were off-

target RNAi nor INF-a/b activated. Instead, choroidal neo-vascularization was blocked by TLR-3 and its adaptor TRIF,

which are localized in various cell types of the cell surface, as

well as the induction of INF-g and interleukin-12.

4.3. Cross-Reactions with the miRNA Pathway

Further undesirable side effects can come about by cross-

reactions with the endogenous miRNA pathway. As

explained in the Introduction, siRNAs and miRNAs function

by very similar mechanisms. For this reason it is hardly

surprising that siRNAs can act as miRNAs. This means that

siRNAs can interact with the 3-UTR of mRNAs by partial

homology and can inhibit their translation without triggering

their degradation.[106,107]

Furthermore, expressed shRNAs can block the endoge-nous miRNA pathway. A pronounced liver toxicity was

observed after a high dose of viral vectors carrying an shRNA

expression cassette was injected into mice.[108] Of the 49

shRNAs tested, 36 caused liver damage, which in 23 cases was

fatal. Presumably, the cellular miRNA pathway was disturbed

by, among other things, over-saturation of Exportin-5, which

is responsible for transporting miRNA precursors out of thenucleus and into the cytoplasm. No side effects were observed

at a lower concentration of the vector, in contrast, and

protection from HBV was achieved in an animal model for up

to a year. In response to this work, a recently published study

investigated whether chemically synthesized siRNAs have an

influence on cellular miRNAs.[109] Liposomal delivery of the

siRNAs resulted in the expression of hepatocyte-specific

genes being inhibited by around 80%. The level and the

function of several investigated miRNAs were not influenced

by the siRNA treatment.In conclusion, it is clear that RNAi applications will never

be completely specific. By suitable design of the siRNAs as

well as the use of modified nucleotides, however, the

unspecific effects can be minimized. In addition, the dose of

the siRNAs or shRNAs should be as low as possible. The

reliability of the results of functional analyses can be

increased by verifying the phenotype with multiple independ-

ent siRNAs. For therapeutic applications, it must be remem-

bered that small molecular substances usually also have

numerous (toxic) side effects. For this reason, the same safety

standards should apply to the preclinical development of

RNAi applications as for other substances.

5. Delivery

Oligonucleotides are multiply negatively charged macro-

molecules which cross the hydrophobic cell membrane with

difficulty. The delivery of the siRNAs into cells presents one

of the greatest challenges to the development of RNAi

applications. For cell-culture applications, transfection

reagents are commonly used, which often have toxic sideeffects in animals or humans. Previous work from the

antisense field established that a certain amount of oligonu-

cleotides are spontaneously taken up by cells in vivo. Thus,

siRNAs also work without a carrier when locally applied, suchas through intranasal delivery[110] or intrathecal injection.[24] It

should be remembered in this case that local application can

create a high concentration of the siRNA in a spatially

restricted area. Additional measures are required for efficient

systemic delivery. Basically, the approaches can be divided

into nonviral delivery of chemically synthesized siRNAs and

viral delivery of shRNA expression cassettes. The preferred

method depends on the application: for temporary diseases

such as acute infections, the short-acting siRNAs can be

sufficient, while for chronic diseases, such as HIV infection or

metabolic diseases, the vector-based method is presumably

more advantageous to avoid repeated dosing.

Figure 6. Toll-like Receptors (TLR). A) Signaling pathway of the TLRs.B) Cellular localization and ligands which activate the various TLRs.LPS: Lipopolysaccharide; CpG: cytidine-phosphate-guanosine; MyD88:myeloid differentiation primary response protein 88; NF-kB: nuclearfactor kappa beta; MAPK: mitogen-activated protein kinases; IRF:interferon regulatory factor. (Figure modified from Ref. [104].)


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5.1. Nonviral Delivery

In the first in vivo applications of RNAi free siRNAs were

applied by hydrodynamic injection in to the tail vein. [111] Thisinvolves injection of a relatively large volume of the siRNA

solution in a short time at high pressure. In this way the

siRNAs are preferentially taken up by the liver, and proof of

principle was demonstrated in practice by the knockdown of

target genes in this target organ. This method is, however,

very harsh and not viable for humans. For this reason,

intensive work on the development of biocompatible proce-dures for the delivery of siRNAs has been going on for many

years.

In vivo systemic application of siRNAs requires that they

overcome numerous barriers to unfold their activity:[112] Free

oligonucleotides are rapidly filtered from the blood by the

kidneys and subsequently excreted. In addition, unmodified

siRNAs are rapidly degraded by nucleases (see Section 2.2),

and foreign macromolecules are phagocytized by the reticulo-

endothelial system and deposited in the liver and spleen. The

half-life of the siRNAs in the bloodstream can be extended byhydrophobic polymers such as polyethylene glycol (PEG).

The siRNAs must then overcome the capillary endothelium

and diffuse into the extracellular matrix of the target tissue.

Uptake into the cells normally occurs by endocytosis, during

which an important step is the release of the siRNA from the

endosomes into the cytoplasm, where they manifest their

activity. There are numerous methods to aid these processes,

of which the most important will be discussed here.

5.1.1. Unspecific Delivery

Many substances are packed into liposomes to improve

their pharmacokinetic characteristics. Liposomes form aphospholipid bilayer surrounding an aqueous compartment,

in which polar substances can be stored, and mediate uptakeof the substances into the cells by some form of vesicular

transport, for example, through endosomes. Cationic lipids

are particularly well suited for the delivery of negatively

charged nucleic acids. Most commercially available trans-

fection reagents form lipoplexes with the oligonucleotides; in

these lipoplexes the siRNA is not contained in the inner

compartment. However, numerous new, less-toxic formula-tions have been developed for in vivo applications. Usually

lipoplexes and liposomes are surrounded by PEG (Fig-

ure 7A,B) to achieve longer circulation in the blood stream

and to reduce the toxicity. In addition, fusogenic lipids can beadded, which improve the release of the siRNAs from the

endosomes. While free siRNAs are rapidly excreted by the

kidneys after intravenous injection, an siRNA labeled with

the fluorescent dye Cy3 that was administered as an siRNA

lipoplex could be detected in many organs.[65] The siRNAs

remained, unfortunately, primarily in the endothelial cells of

the blood vessels and, therefore, barely penetrated into the

tissues themselves.

Liposomal delivery of the completely modified siRNA

against HBV described in Section 2.2 increased both the

efficiency and duration of action in a mouse model. [113] The

siRNA was encapsulated in stable nucleic acid-lipid particles

(SNALPs), which consist of a cationic and a fusogenic lipid

and are also PEGylated (Figure 7B). SNALPs were subse-

quently used to test an siRNA against apolipoprotein B inprimates.[25] The level of the mRNA in the liver was reduced

by more than 90 % after a single injection, and as a result the

protein, the serum cholesterol, and the level of low-density

lipoprotein (LDL) was reduced. This showed that liposome-

mediated siRNA delivery could be successfully tested in a

clinically relevant context. The knockdown of apolipopro-

tein B demonstrates two further aspects: First, a partial

reduction of the target protein is sufficient to reach a relevant

therapeutic benefit, namely reduction of LDL to a normal

level. For the use of RNAi against tumors or viral infections,

however, the greatest possible knockdown of the target gene

must be reached to prevent a relapse of the disease. Secondly,

Figure 7. Nonviral delivery of siRNAs. A) Lipoplex: cationic lipids(gray) form complexes with the negatively charged siRNAs (red). PEG(yellow) is frequently attached to improve the pharmacokinetic charac-teristics. B) Liposomes in which the cationic lipids enclose the siRNA.C) siRNA coupled to cholesterol to increase its lipophilicity. D) Specificdelivery by coupling of siRNAs on the antigen-binding fragment of anantibody through positively charged protamine. E) Direct coupling ofan siRNA to an aptamer for tumor-cell-specific delivery. F) Neuronaldelivery by a peptide of the rabies virus glycoprotein (RVG) with anarginine nonamer (9R) at its carboxy terminus to bind the siRNA.G) Receptor-mediated delivery by coupling of a ligand (F: Folate) to aDNA oligonucleotide (blue), that hybridizes with siRNA (sense strand:green, antisense strand: red). Further details are described in the text.

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the advantages of RNAi technology lies in the fact that any

chosen gene can be inhibited, not just the so-called drugable

targets, against which traditional small-molecule substances

can be directed.Besides the lipid-based systems, various other polymers

have been employed for the delivery of siRNAs. One of the

most intensively investigated polymers for the delivery of

nucleic acids is polyethyleneimine (PEI). The linear or

branched PEI polymers are strongly positively charged and

can therefore form complexes with siRNAs and electrostati-

cally interact with the cell surface. The complexes are takenup by endocytosis, and PEI improves the release of the siRNA

by destroying the endosomes. PEI-siRNA complexes can be

employed successfully to limit influenza infections in mice, for

example.[114]

Nanoparticles from completely different substances have

also been developed. For example, Medarova et al. used

nanoparticles, which after systemic application allow the

delivery and proof of siRNA uptake at the same time.[115] The

samples consisted of magnetic nanoparticles labeled with a

dye which absorbs in the near-infrared region so thataccumulation in tumors could be observed by magnetic

resonance imaging (MRI) and near-infrared in vivo optical

imaging (NIRF). The nanoparticles were equipped with a

myristoyl-coupled polyarginine peptide for translocation

across the membrane. In an alternative system, carbon fiber

nanotubes were used, which facilitated entry of siRNAs into

T cells and primary cells, which are otherwise difficult to

transfect with liposomal systems.[116]

An alternative strategy is to couple lipophilic molecules

directly to the siRNA. Of a series of tested groups, cholesterol

and 12-hydroxylauric acid coupled to the 3 end of the sense

strand proved to be the best suited to ensure efficient uptake

by the cells and knockdown of the target gene.[60]

As a result, acholersterol-coupled siRNA (Figure 7 C) was injected into

the tail veins of mice.[117] Cellular uptake and silencing of the

target protein (apolipoprotein B) could be shown in the liver

and the jejunum (a section of the small intestine).

In addition to lipophilic groups, cell-penetrating peptides

(CPP) can improve the cellular uptake of oligonucleotides.[118]

Interestingly, phosphorothioate oligonucleotides which are

not covalently attached to an siRNA improve the uptake by a

caveolin-mediated mechanism.[119] This resulted in the expres-sion of lamin in primary HUVEC endothelial cells being

inhibited.

5.1.2. Cell-Type-Specific Delivery

The development of systems that allow specific delivery of

siRNAs to their target cells represents a great advance. This

approach allows the applied doses to be smaller and possible

side effects in other tissues can be avoided. An elegant

possibility consists of coupling the siRNA to an antibody

which recognizes a protein on the surface of special cells. In a

ground-breaking study, the antigen-binding fragment of an

antibody against the HIV glycoprotein, which was fused to

protamine, was used (Figure 7 D).[120] This positively charged

protein can assemble with approximately six (negatively

charged) siRNAs in a noncovalent manner. With this

construct it was possible to inhibit an HIV infection of

primary T cells, which are difficult to transfect with lipid-

based strategies. The authors succeeded in vivo with their

antibody strategy in delivering the siRNAs to tumor cellswhich presented the ligand proteins of the antibodies on their

cell surface.

To avoid the need to combine two classes of molecules

(proteins and nucleic acids) siRNAs have been coupled to

aptamersligand-binding, in vitro selected nucleic acids. In a

first effort, a streptavadin bridge was used to bind an siRNA

against lamin to an aptamer against the prostate-specificmembrane antigen (PSMA),[121] a membrane receptor which

is expressed in prostate cancer cells and the vascular

endothelia of tumors. This conjugate made efficient silencing

possible, but is relatively complex because of the biotin

streptavadin bridge. For this reason, the siRNA was coupled

directly to a different aptamer against PSMA in a further

study (Figure 7 E).[122] Once in the cell, the siRNA is removed

from the aptamer by Dicer. In an animal model it was possible

to inhibit the growth of a tumor from human prostate

carcinoma cells with an aptamer-coupled siRNA against Plk1.The treatment of neurological diseases is complicated by

the need to pass through the bloodbrain barrier, which often

prevents the entry of drugs from the bloodstream into the

brain. To overcome this barrier, a 29 amino acid long peptide

from the rabies virus glycoprotein was coupled with an

arginine nonamer to an siRNA (Figure 7F).[123] The peptide

bound to the acetylcholine receptor, which is expressed by

neuronal cells, so that the conjugate specifically penetrates

neurons. In vivo, an intravenously injected siRNA with the

peptide succeeded in getting into brain cells, and protected

mice from an infection with Japanese encephalitis virus.

A further possibility for cell-type-specific delivery is the

coupling of a receptor ligand (such as folate) to a DNAoligonucleotide (Figure 7 G).[124] This DNA oligonucleotide

hybridizes with the 3 extended end of the antisense strand of

the siRNA, and the ligand mediates the uptake into the cells

of the construct, which consists of two RNA molecules and a

DNA oligonucleotide. Presumably, Dicer or an RNase H then

produces the mature siRNA.

Further details concerning nonviral delivery of siRNAs

are described in recently published reviews.[112,125127]

5.2. Viral Delivery

Viruses belong to the most dangerous pathogens forhumans, and therapies against them are often inadequate or

not available. For the last 20 years, however, a concept has

been pursued to introduce therapeutically useful genes into

patients with the help of viral vectors. In the process, the

viruses are usually changed such that essential components

for replication are missing so that they cannot produce

progeny viruses and therefore cannot harm the patient or

others (Figure 8). Although worldwide over 220 genes have

been transferred in almost 1500 clinical trials,[128] the real

breakthrough in gene therapy has not yet been accomplished.

High hopes were placed on combining the new and very

efficient RNAi technology with the experience of gene


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therapy.[90,129] This involves getting the shRNA expressioncassettes into the cells by means of viral vectors. This form of

gene transfer is usually significantly more efficient than the

nonviral delivery of siRNAs. Three types of vectors are

primarily used: Retroviral vectors, adenoviral vectors, as wellas vectors based on the adeno-associated virus (AAV). The

most important advantages and disadvantages of the three

vector types are summarized in

Table 1. Past experience has

shown that it is impossible to find

a vector which is optimal for all

indications, instead the choice of

vector type depends on the

intended specific therapeutic use.

5.2.1. Retroviral Vectors

Retroviruses have an RNA genome, which is copied into a

double-stranded DNA, which in turn is integrated into thehost genome as a proviral DNA. This characteristic is

maintained in therapeutically used retroviral vectors so as

to permanently express the transgene. The immune response

to these vectors is weak and the viruses are modified such that

they can no longer leave the cells and cause damage.

The family of the Retroviridae are divided into the

subgroups onco-retroviruses and lentiviruses. Onco-retrovi-ruses can only transduce proliferating cells. They are primar-

ily used ex vivo, that is, the cellsfor example hematopoietic

stem cellsare removed from the patient which are trans-

duced with the retrovirus vector in tissue-culture dishes and

are later re-administered to the patient. In this manner

children have been treated who suffer from X-SCID (severe

combined immunodeficiency disorder), which is caused by a

mutation in the gc interleukin receptor gene located on the

X chromosome.[130] The presumed advantage of long-term

expression by stable integration into the host genome provedto be a disadvantage, however, since several of the children

developed leukemia as a consequence of the treatment.[131]

The retroviral vector integrated in the proximity of the LMO2

proto-oncogene promoter and led to the anomalous tran-

scription and expression of LMO2. This finding shows that the

safety of the vectors must be improved; at the same time,

however, one must remember that diseases such as SCID are

frequently untreatable by any other means and lead to the

early death of the affected children.

Lentiviral vectors can, for example, be derived from the

human immunodeficiency virus (HIV). They have the ability

to transduce quiescent as well as proliferating cells, thus

increasing their therapeutic range. Furthermore, their onco-genic potential is presumably less. The G glycoprotein of the

vesicular stomatitis virus can be used as the coat protein for

lentiviral vectors, which allows the transduction of almost any

cell type.

After the demonstration that retorviral vectors are in

principle suited to siRNA-mediated gene silencing by inhib-

iting the reporter gene eGFP,[132] they were employed for

medically relevant purposes. The specific knockdown of the

oncogene K-RasV12 allele in human tumor cells caused themto lose their tumorgenicity.[133] In addition, lentiviral vectors

were used to introduce shRNA expression cassettes against

viruses or their receptors into host cells. A lentivirus vector

proved to be particularly efficient for the inhibition ofhepatitis C virus (HCV). This vector produced several

shRNAs against the virus genome and the host cell receptor

Figure 8. Creation of replication-deficient viral vectors. For gene trans-

fer, the central protein-coding genes of the viral genome are removedand replaced with the transgene or an shRNA expression cassette. Thevector genome is packaged in a packaging cell line expressing the viralgenes, which in most cases are spread over several plasmids. Theresulting virus vector only includes the expression cassette for thetransgene, while the essential virus genes are missing, so that furtherreplication is impossible.

Table 1 : Overview of the most important characteristics of viral vectors.

Retroviral vectors Adenoviral vectors Adeno-associatedvirus vectors

transduction of quiescentcells

noonco-retrovirusyeslentivirus

yes yes

genomic integration yes no no (or limited)potential risks insertional

mutagenesisimmune reaction,cytotoxicity

cytotoxicity

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CD81 at the same time and thereby blocked HCV replication,

CD81 expression, and cell binding of the HCV surface

protein E2.[134] The intensive efforts to use lentiviral vectors

for the transfer of shRNA expression cassettes for thetreatment of HIV infection and an ongoing clinical trial for

this purpose will be described in Section 6.3.2.

5.2.2. Adenoviral Vectors

Adenoviruses have a linear, double-stranded DNA

genome and most often cause respiratory problems inhumans. The genomic DNA of adenoviruses remains episo-

mal in the infected cells, so that no risk of insertional

mutagenesis exists. Unfortunately, they do cause a powerful

immune reaction, which led to a fatal reaction in a clinical

study.[135] Parts of the early genes were removed in the first

generation of adenoviral vectors. The early genes E2 and/or

E4 were deleted in the second generation adenovirus vectors

to further reduce the immunogenicity and to create additional

space for transgenes. In the newest vectors, which are referred

to as gutless, all coding sequences are deleted, so that besidesthe transgene, only the inverted terminal repeats (ITRs) and

the packaging signal Y remain.[136] This approach drastically

reduces the toxicity and immunogenicity of the vectors, and

enables the long-term expression of a transgene.

Adenoviral vectors have already been employed in differ-

ent medical areas for the knockdown of damaging genes. For

example, both guinea pigs and pigs were protected from

infection by foot-and-mouth disease by an shRNA-expressing

adenovirus vector.[137] The adenovirus vector mediated deliv-

ery of shRNA expression cassettes has also been developed

for the treatment of heart diseases. The disruption of the

calcium balance is an important cause of heart failure. With

RNAi-mediated inhibition of phospholamban, an inhibitor ofthe SERCA2A (sarcoplasmic reticulum Ca2+ pump), it was

possible to improve the calcium uptake into the sacroplasmic

reticulum in primary neonatal rat cardiomyocytes.[138]

5.2.3. Vectors Based on Adeno-Associated Virus

Adeno-associated viruses (AAVs) belong to the family of

the Parvoviridae and possesses a comparatively small linear

single-stranded DNA genome. AAVs are attractive vectorsfor gene transfer, since they efficiently transduce target cells

and are nonpathogenic for humans. While natural AAVs

integrate into a specific region in chromosome 19, the genes

required for this are usually deleted from the recombinantvectors, so that they remain primarily episomal. Despite this,

AAV vectors are noteworthy for their long-term, stable

expression of transgenes.

For gene therapeutic uses, the AAV serotype 2 was first

developed as a vector. Since it inefficiently transduced many

cell types such as muscle cells, other serotypes have been used

in the past few years to expand their tropism. The genome of

the AAV-2 vector can be packed in capsids of other serotypes.

This leads to the creation of so-called pseudotype vectors,

with which cells of practically any given tissue can be

transduced.[139] A further disadvantage of the conventional

single-stranded vectorsthe delayed start of the expression

of the transgenecould also be eliminated. Maximal gene

expression is achieved after only a few days with self-

complementary double-stranded AAV vectors.[140]

AAV vectors are intensively employed in RNAi experi-ments because of their numerous advantages. For example,

the dopamine-synthesizing enzyme tyrosine hydroxylase was

down-regulated in the midbrain neurons of mice with

shRNA-expressing AAV vectors in one of the first in vivo

studies.[141] As a result, behavioral changes such as a motor-

performance deficit and altered reaction to a psychostimulant

were seen. The faster acting self-complementary AAV vectorsproved useful for cell-culture experiments: the mRNA of the

target gene was reduced by up to 80% after transduction of a

culture of rat-lung fibroblasts for 72 h.[142]

6. Applications of RNA Interference

6.1. Investigation of Gene Function

The sequencing of the human genome as well as those ofmany other eukaryotic model organisms rates as one of the

most important developments of the last few decades in the

life sciences. In many cases, however, only the sequence is

known, while the function of the coded protein remains

unknown. Determination of gene function has become one of

the most important tasks of present research. At about the

same time as the completion of the major sequencing projects,

RNAi was established as a method to allow the creation of

loss-of-function phenotypes in a comparatively rapid and

simple manner. This led to the adoption in only a few years of

RNAi as a standard method of molecular biological research

that is employed in a very large number of biochemical

laboratories.Since silencing is based on the pairing between the mRNA

of the gene of interest and the siRNA guide strand, genefunctions can be investigated significantly faster than they can

be with small-molecule inhibitors, which must first be

identified in laborious high-throughput screens. In addition,

closely related isoforms of proteins can be selectively turned

off by suitable selection of target sequences to investigate

their specific functions,[143] which is almost never possible with

pharmacological substances. Even when the goal of apharmaceutical project is the development of a traditional

drug, RNAi offers a rapid method to validate the target. [144]

The unspecific effects of RNAi applications discussed in

Section 4 must, however, be kept in mind; thus controls werealready laid down with which the specificity of an RNAi

experiment should be proven in the early stages of the

research.[145] These include, among other things, suitable

controls of the knockdown at the mRNA and protein levels,

doseresponse curves of the siRNA, as well as the use of

multiple siRNAs against the same target.

6.2. Screens with Genome-Wide Libraries

Besides the analysis of the function of individual genes,

many genes can also be investigated at the same time by using


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siRNA libraries. In a first example, every member of the

family of de-ubiquitinating enzymes was selectively turned off

with shRNAs.[146] This approach led to the discovery that the

knockdown of familial cylindromatosis tumor suppressor(CYLD) led to an increase in the activity of the transcription

factor NF-kB after TNF-a stimulation (Figure 9). Interest-

ingly, the activation could be prevented by an aspirin

derivative. As a result, patients with cylindromatosis, mostly

benign tumors of the skin appendages, were treated with

salicylic acid, which in some cases led to a full remission.[147]

This example illustrates how RNAi has led to new indications

for well-known drugs.A number of libraries of siRNAs, endoribonuclease-

prepared siRNAs, and shRNA-expressing retrovirus vectors

have now been developed which cover the entire human

genome. Genome-wide screens are primarily used in viro-

logical or oncological studies. In this way over 250 cellular

factors necessary for HIV-1 infection could be identified in a

comprehensive screen with 4 siRNAs against each of the

approximately 21000 human genes.[148] This led not only to

additional information about the viral life cycle but alsoidentified new potential therapeutic targets. In a screen of

retroviral vectors with miRNA-type shRNAs against around

3000 genes, proteins were identi-

fied that are involved in the pro-liferation of cancer cells.[149] In a

further genome-wide screen,

potential tumor suppressors were

found which were required to

block the proliferation of fibro-

blasts and melanocytes that con-

tained an activated mutant of the

braf proto-oncogene.[150]

6.3. Therapeutic Applications

The decades-long experience with the clinical develop-

ment of antisense oligonucleotides[151] and ribozymes[152] wasutilized in the therapeutic application of siRNAs. Only this

can explain how the first RNAi treatments were started on

humans just three and a half years after siRNAs were first

used in mammalian cells. Antisense oligonucleotides and

siRNAs differ from conventional substances by their size, and

large-scale synthesis of these oligomers causes considerable

difficulties and high costs. Furthermore, the two strands of thesiRNAs must be synthesized separately and subsequently

hybridized. This process has to guarantee the formation of a

uniform drug that must, in the end, satisfy the requirements of

the regulatory authorities. Local application was selected for

the first proof-of-concept studies because of the previously

discussed delivery problems. Table 2 shows the most

advanced, RNAi-based clinical trails.

6.3.1. Eye Diseases

The eye is a spatially well-defined organ with low nuclease

activity in which the active agent can be injected intravitreally

(directly into the vitreous body) comparatively easily. The

only two oligonucleotides which have been approved by the

American Food and Drug Administration (FDA) are for the

treatment of eye diseases. The antisense oligonucleotide

Fomivirsen is directed against cytomegalovirus, which causes

retinitis in AIDS patients; Macugen is an aptamer for the

treatment of age-related macular degeneration (AMD), one

of the most common eye diseases among the elderly. The first

RNAi-based clinical studies were started at the end of 2004

with an siRNA against VEGF. Inhibiting the expression of

VEGF should block neovascularization in patients withAMD. The siRNA has since been tested under the name

Bevasiranib in a phase III trial by the company Opko Health.Sirna Therapeutics (since bought by Merck & Co. Inc.,

USA) initiated the first clinical studies with a chemically

modified siRNA. The siRNA with the code Sirna-027 was

stabilized by unpaired deoxythymidine with a phosphoro-

thioate bond and inverted abasic sugar residues on the ends of

the antisense and sense strand, respectively, and is directed

against the VEGF receptor-1. This approach also enabled thetreatment of patients with AMD. An intravitreal injection of

the siRNA reduced the area of neovascularization by as much

Figure 9. Function of the tumor suppressor CYLD, which was identi-fied by means of an RNAi screen. CYLD works as an inhibitor in theNF-kB signaling pathway. The loss of the CYLD function leads touncontrolled growth. The pathway can also be inhibited by usingsodium salicylate or prostaglandin-1 (PGA1). TRAF: TNF-receptor-associated factor; IKK: IkB kinase complex. Scheme adapted fromRef. [146].

Table 2: RNAi in clinical trials (based on Ref. [153]).

Company Disease Product Status

Sirna/MERCK AMD Sirna-027/AGN211745 phase IIQuark Pharma. (Pfizer) AMD RTP801i-14 phase I/IIOpko Health AMD Bevasiranib phase IIIBenitec/City of Hope HIV/AID

2009 kur reck

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